A non-coding indel polymorphism in the fruitless gene of Drosophila melanogaster exhibits antagonistically pleiotropic fitness effects

The amount of genetic variation for fitness within populations tends to exceed that expected under mutation–selection–drift balance. Several mechanisms have been proposed to actively maintain polymorphism and account for this discrepancy, including antagonistic pleiotropy (AP), where allelic variants have opposing effects on different components of fitness. Here, we identify a non-coding indel polymorphism in the fruitless gene of Drosophila melanogaster and measure survival and reproductive components of fitness in males and females of replicate lines carrying each respective allele. Expressing the fruitless region in a hemizygous state reveals a pattern of AP, with one allele generating greater reproductive fitness and the other conferring greater survival to adulthood. Different fitness effects were observed in an alternative genetic background, which may reflect dominance reversal and/or epistasis. Our findings link sequence-level variation at a single locus with complex effects on a range of fitness components, thus helping to explain the maintenance of genetic variation for fitness. Transcription factors, such as fruitless, may be prime candidates for targets of balancing selection since they interact with multiple target loci and their associated phenotypic effect

Identifying SARS-CoV-2 antiviral compounds by screening for small molecule inhibitors of nsp13 helicase

The coronavirus disease 2019 (COVID-19) pandemic, which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a global public health challenge. While the efficacy of vaccines against emerging and future virus variants remains unclear, there is a need for therapeutics. Repurposing existing drugs represents a promising and potentially rapid opportunity to find novel antivirals against SARS-CoV-2. The virus encodes at least nine enzymatic activities that are potential drug targets. Here, we have expressed, purified and developed enzymatic assays for SARS-CoV-2 nsp13 helicase, a viral replication protein that is essential for the coronavirus life cycle. We screened a custom chemical library of over 5000 previously characterized pharmaceuticals for nsp13 inhibitors using a fluorescence resonance energy transfer-based high-throughput screening approach. From this, we have identified FPA-124 and several suramin-related compounds as novel inhibitors of nsp13 helicase activity in vitro. We describe the efficacy of these drugs using assays we developed to monitor SARS-CoV-2 growth in Vero E6 cells

The Internet as a Small Business E-Commerce Ecosystem.

The purpose of this chapter is to analyse how the ecosystem concept can be applied to small businesses and how the Internet and e-commerce can help SMEs harness the required resources to enhance their competitive performance in the marketplace. The chapter will investigate the wide variety of e-commerce applications that are available to small businesses to help address the issue of limited resources. It will provide an ecosystem map illustrating how each functional area of a small business can utilise Internet e-commerce applications to enhance their resource base. The chapter also explores the opportunities and threats that the e-commerce ecosystem model poses for small, medium-sized enterprises (SMEs). This is based upon empirical research consisting of three focus group interviews undertaken with small and medium-sized retail service firms located in the Herefordshire and Worcestershire regions of the United Kingdom in January–February 2014

Identifying SARS-CoV-2 antiviral compounds by screening for small molecule inhibitors of Nsp3 papain-like protease

The COVID-19 pandemic has emerged as the biggest life-threatening disease of this century. Whilst vaccination should provide a long-term solution, this is pitted against the constant threat of mutations in the virus rendering the current vaccines less effective. Consequently, small molecule antiviral agents would be extremely useful to complement the vaccination program. The causative agent of COVID-19 is a novel coronavirus, SARS-CoV-2, which encodes at least nine enzymatic activities that all have drug targeting potential. The papain-like protease (PLpro) contained in the nsp3 protein generates viral non-structural proteins from a polyprotein precursor, and cleaves ubiquitin and ISG protein conjugates. Here we describe the expression and purification of PLpro. We developed a protease assay that was used to screen a custom compound library from which we identified dihydrotanshinone I and Ro 08-2750 as compounds that inhibit PLpro in protease and isopeptidase assays and also inhibit viral replication in cell culture-based assays

Break dosage, cell cycle stage and DNA replication influence DNA double strand break response

DNA double strand breaks (DSBs) can be repaired by non-homologous end joining (NHEJ) or homology-directed repair (HR). HR requires nucleolytic degradation of 5′ DNA ends to generate tracts of single-stranded DNA (ssDNA), which are also important for the activation of DNA damage checkpoints. Here we describe a quantitative analysis of DSB processing in the budding yeast Saccharomyces cerevisiae. We show that resection of an HO endonuclease-induced DSB is less extensive than previously estimated and provide evidence for significant instability of the 3′ ssDNA tails. We show that both DSB resection and checkpoint activation are dose-dependent, especially during the G1 phase of the cell cycle. During G1, processing near the break is inhibited by competition with NHEJ, but extensive resection is regulated by an NHEJ-independent mechanism. DSB processing and checkpoint activation are more efficient in G2/M than in G1 phase, but are most efficient at breaks encountered by DNA replication forks during S phase. Our findings identify unexpected complexity of DSB processing and its regulation, and provide a framework for further mechanistic insights

Diversity of Eukaryotic DNA Replication Origins Revealed by Genome-Wide Analysis of Chromatin Structure

Eukaryotic DNA replication origins differ both in their efficiency and in the characteristic time during S phase when they become active. The biological basis for these differences remains unknown, but they could be a consequence of chromatin structure. The availability of genome-wide maps of nucleosome positions has led to an explosion of information about how nucleosomes are assembled at transcription start sites, but no similar maps exist for DNA replication origins. Here we combine high-resolution genome-wide nucleosome maps with comprehensive annotations of DNA replication origins to identify patterns of nucleosome occupancy at eukaryotic replication origins. On average, replication origins contain a nucleosome depleted region centered next to the ACS element, flanked on both sides by arrays of well-positioned nucleosomes. Our analysis identified DNA sequence properties that correlate with nucleosome occupancy at replication origins genome-wide and that are correlated with the nucleosome-depleted region. Clustering analysis of all annotated replication origins revealed a surprising diversity of nucleosome occupancy patterns. We provide evidence that the origin recognition complex, which binds to the origin, acts as a barrier element to position and phase nucleosomes on both sides of the origin. Finally, analysis of chromatin reconstituted in vitro reveals that origins are inherently nucleosome depleted. Together our data provide a comprehensive, genome-wide view of chromatin structure at replication origins and suggest a model of nucleosome positioning at replication origins in which the underlying sequence occludes nucleosomes to permit binding of the origin recognition complex, which then (likely in concert with nucleosome modifiers and remodelers) positions nucleosomes adjacent to the origin to promote replication origin function

Identifying SARS-CoV-2 antiviral compounds by screening for small molecule inhibitors of Nsp5 main protease

The coronavirus 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), spread around the world with unprecedented health and socio-economic effects for the global population. While different vaccines are now being made available, very few antiviral drugs have been approved. The main viral protease (nsp5) of SARS-CoV-2 provides an excellent target for antivirals, due to its essential and conserved function in the viral replication cycle. We have expressed, purified and developed assays for nsp5 protease activity. We screened the nsp5 protease against a custom chemical library of over 5000 characterised pharmaceuticals. We identified calpain inhibitor I and three different peptidyl fluoromethylketones (FMK) as inhibitors of nsp5 activity in vitro, with IC(50) values in the low micromolar range. By altering the sequence of our peptidomimetic FMK inhibitors to better mimic the substrate sequence of nsp5, we generated an inhibitor with a subnanomolar IC(50). Calpain inhibitor I inhibited viral infection in monkey-derived Vero E6 cells, with an EC(50) in the low micromolar range. The most potent and commercially available peptidyl-FMK compound inhibited viral growth in Vero E6 cells to some extent, while our custom peptidyl FMK inhibitor offered a marked antiviral improvement

Computational Methods to Study Kinetics of DNA Replication

New technologies such as DNA combing have led to the availability of large quanti-ties of data that describe the state of DNA while undergoing replication in S phase. In this chapter, we describe methods used to extract various parameters of replica-tion — fork velocity, origin initiation rate, fork density, numbers of potential and utilized origins — from such data. We first present a version of the technique that applies to “ideal ” data. We then show how to deal with a number of real-world complications, such as the asynchrony of starting times of a population of cells, the finite length of fragments used in the analysis, and the finite amount of DNA in a chromosome. Key words: DNA replication, replication fork velocity, origin initiation

CRK9 contributes to regulation of mitosis and cytokinesis in the procyclic form of Trypanosoma brucei

<p>Abstract</p> <p>Background</p> <p>The <it>Trypanosoma brucei </it>cell cycle is regulated by combinations of cyclin/CRKs (cdc2 related kinases). Recently, two additional cyclins (CYC10, CYC11) and six new CRK (CRK7-12) homologues were identified in the <it>T. brucei </it>genome database <abbrgrp><abbr bid="B1">1</abbr><abbr bid="B2">2</abbr></abbrgrp>.</p> <p>Results</p> <p>Individual RNAi knockdowns of these new proteins in the procyclic form of <it>T. brucei </it>showed no apparent phenotype except for the CRK9 depletion, which enriched the cells in G2/M phase. But a similar CRK9 knockdown in the bloodstream form caused no apparent phenotype. CRK9 lacks the typical PSTAIRE motif for cyclin binding and the phenylalanine "gatekeeper" but binds to cyclin B2 <it>in vitro </it>and localizes to the nucleus in both forms of <it>T. brucei</it>. CRK9-depleted procyclic-form generated no detectable anucleate cells, suggesting an inhibition of cytokinesis by CRK9 depletion as well. The knockdown enriched cells with one nucleus, one kinetoplast and two closely associated basal bodies with an average distance of 1.08 mm in between, which was shorter than the control value of 1.36 μm, and the cells became morphologically deformed and rounded with time.</p> <p>Conclusion</p> <p>CRK9 may play a role in mediating the segregation between the two kinetoplast/basal body pairs prior to cytokinetic initiation. Since such a segregation over a relatively significant distance is essential for cytokinetic initiation only in the procyclic but may not be in the bloodstream form, CRK9 could be specifically involved in regulating cytokinetic initiation in the procyclic form of <it>T. brucei</it>.</p

An Increase in Mitochondrial DNA Promotes Nuclear DNA Replication in Yeast

Coordination between cellular metabolism and DNA replication determines when cells initiate division. It has been assumed that metabolism only plays a permissive role in cell division. While blocking metabolism arrests cell division, it is not known whether an up-regulation of metabolic reactions accelerates cell cycle transitions. Here, we show that increasing the amount of mitochondrial DNA accelerates overall cell proliferation and promotes nuclear DNA replication, in a nutrient-dependent manner. The Sir2p NAD+-dependent de-acetylase antagonizes this mitochondrial role. We found that cells with increased mitochondrial DNA have reduced Sir2p levels bound at origins of DNA replication in the nucleus, accompanied with increased levels of K9, K14-acetylated histone H3 at those origins. Our results demonstrate an active role of mitochondrial processes in the control of cell division. They also suggest that cellular metabolism may impact on chromatin modifications to regulate the activity of origins of DNA replication